Promiscuous G-protein compositions and their use

ABSTRACT

Disclosed are compositions and methods for their use, such as in identifying G-protein coupled receptors and ligands and compounds that modulate signal transduction. The compositions and methods employ promicuous G-proteins. Activation of the promiscous G-protein can be detected in a variety of assays, including assays in which activation is indicated by a change in fluorescence emission of a sample that contains the composition.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. Section 119 toprovisional patent application 60/020,234 filed on Jun. 21, 1996, byNegulescu et al., which is herein incorporated by reference and of whichthis application is a continuation in part.

FIELD OF THE INVENTION

The invention relates to compositions and methods for identifyingG-protein coupled receptors (GPCRs) and compounds that modulate activityof G-proteins or their receptors.

BACKGROUND

Many physiological signals (e.g., sensory, hormonal and neurotransmittersignals) are transduced from extracellular to intracellular environmentsby cell surface receptors termed G-protein coupled receptors (GPCRs)(for a review, see Neer, 1995, Cell 80:249-257). Typically, GPCRscontain seven transmembrane domains. Putative GPCRs can be identified onthe basis of sequence homology to known GPCRs.

GPCRs mediate signal transduction across a cell membrane upon thebinding of a ligand to an extracellular portion of a GPCR. Theintracellular portion of a GPCR interacts with a G-protein to modulatesignal transduction from outside to inside a cell. A GPCR is thereforesaid to be “coupled” to a G-protein. G-proteins are composed of threepolypeptide subunits: an α subunit, which binds and hydolyzes GTP, and adimeric βγ subunit. In the basal, inactive state, the G-protein existsas a heterotrimer of the α and βγ subunits. When the G-protein isinactive, guanosine diphosphate (GDP) is associated with the α subunitof the G-protein. When a GPCR is bound and activated by a ligand, theGPCR binds to the G-protein heterotrimer and decreases the affinity ofthe Gα subunit for GDP. In its active state, the G submit exchanges GDPfor guanine triphosphate (GTP) and active Gα subunit disassociates fromboth the receptor and the dimeric βγ subunit. The disassociated, activeGα subunit transduces signals to effectors that are “downstream” in theG-protein signalling pathway within the cell. Eventually, theG-protein's endogenous GTPase activity returns active G subunit to itsinactive state, in which it is associated with GDP and the dimeric βγsubunit.

Numerous members of the heterotrimeric G-protein family have beencloned, including more than 20 genes encoding various Gα subunits. Thevarious G subunits have been categorized into four families, on thebasis of amino acid sequences and functional homology. These fourfamilies are termed Gα_(s), Gα_(i), Gα_(q), and Gα₁₂. Functionally,these four families differ with respect to the intracellular signalingpathways that they activate and the GPCR to which they couple.

For example, certain GPCRs normally couple with Gα_(s). and, throughGα_(s), these GPCRs stimulate adenylyl cyclase activity. Other GPCRsnormally couple with Gα_(q), and through Gα_(q), these GPCRs canactivate phospholipase C (PLC), such as the β isoform of phospholipase C(PLCβ) (Stemweis and Smrcka, 1992, Trends in Biochem. Sci. 17:502-506).

Certain G-proteins are considered “promiscuous” G-proteins because theirG subunits allow them to couple with GPCRs that normally couple withG-proteins of other families. For example, two members of the Gα_(q)family, human Gα₁₆ and its murine homolog Gα₁₅, have been shown intransient cell-based systems to possess promiscuous receptor coupling.Although G-proteins having these G subunits are promiscuous with respectto the GPCR with which they couple, these G-proteins retain the abilityto couple with a specific downstream effector. In other words,regardless of which receptor is used to activate these G-proteins, theactive promiscuous G subunit nonetheless activates PLCβ.

SUMMARY OF THE INVENTION

The invention provides for the first time, a stable, isolated cell thatexpresses, from a construct, a Gα subunit of a promiscuous G-protein(e.g., Gα₁₅, or Gα₁₆). In a preferred embodiment, a polynucleotideencoding a promiscuous G subunit is linked to an inducible promoter onthe construct. To detect activation of the promiscuous G-protein, thecell can include an additional construct that includes a reporter geneoperably linked to a promoter that is activated (usually indirectly) byan active G subunit of a promiscuous G-protein. For the first time,these cells allow occupation of any G-protein coupled receptor (GPCR) bya ligand to be detected using a signal transduction detection system,such as expression of a reporter gene. Other signal transductiondetection systems include detecting changes in intracellular activity,such as methods of detecting G-protein activation from changes incalcium levels in the cell. Preferred methods for detecting expressionof the reporter gene involve detecting a change in fluorescence emissionfrom a sample that includes the cell containing the reporter gene.

Another key aspect of the invention is functional selection of stablecell lines. Stable cell lines can be functionally selected using asignal transduction detection system as described herein. Stable cellsare generated that tolerate the expression of a target protein (such asan ion channel, kinase, phospholipase, phosphatase, transcriptionfactors or GPCR) or a signal transduction coupling protein (e.g. Gprotein) or both.

The cells of the invention can be employed in methods for (i)determining whether a polypeptide is a GPCR for a given ligand; (ii)determining whether a “test” ligand is a ligand for a given GPCR; (iii)functionally characterizing the ability of a ligand to activate variousGPCRs; and (iv) determining whether a compound modulates signaltransduction in a cell (e.g., as an agonist or antagonist).

Another aspect of the invention includes, a method of a identifying of aligand for a GPCR, the method comprising:

-   -   a) contacting a cell with a test chemical, wherein said cell is        expressing a GPCR and arises from a cell line subjected to        functional cell analysis with a signal transduction detection        system; and    -   b) detecting a signal with a signal transduction detection        system.

A related aspect of the invention includes, a method for identifyingmodulators of signal transduction in a cell, the method comprising:

-   -   c) contacting a cell with a compound that directly or indirectly        activates a Gα protein encoded by a polynucleotide, wherein said        cell arises from a cell line subjected to functional cell        analysis with a signal transduction detection system,    -   d) contacting said cell with a test chemical, and    -   e) detecting a signal with a signal transduction detection        system. The invention also includes, a method for identifying a        GPCR for a given ligand or method of identifying a modulator of        a GPCR, the method comprising:    -   f) expressing a putative GPCR or a GPCR of known function in a        cell, wherein said cell arises from a cell line subjected to        functional cell analysis with a signal transduction detection        system;    -   g) contacting contacting said cell with a test chemical or a        ligand known to be a GPCR ligand; and    -   h) detecting a calcium level within said cell.

Also included within the invention, are kits that components for signaltransduction detection systems and cells of the invention.

The invention also includes methods of identifying modulators that donot employ a GPCR, but which employ direct activators of G-proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows uses of promiscuous Gα-protein to detect activation of avariety of GPCRs. Three major classes of GPCRs are diagrammed couplingto their endogenous signaling cascade. Promiscuous G-protein expressionwill allow various classes to couple to the PLC cascade.

FIG. 2 shows one embodiment of the invention that can be used forscreening for Gq type G-protein activation. Modulation of GPCR activityinitiates signaling cascade via PLC. PLC signals can be detected usingNFAT responsive element linked to a transcriptional readout (e.g.reporter gene).

FIG. 3 is a copy of a photograph of a Western blot. Lanes correspondingto samples that lacked or contained doxycyclin are indicated by “−” and“+” respectively. Lanes 1-4 show inducible expression of Gα₁₅ in twodistinct clones of COS-7 cells. Lanes 5-8 show inducible expression ofGα₁₆ in two distinct clones. Gα₁₅ and Gα₁₆ each appear as a specieshaving a molecular weight of approximately 43 kDA in each of the “+”lanes. As a negative control, COS-7 cells were analyzed in lane 9. Thepredicted molecular weight of Gα subunit is 43-45 kDa

FIG. 4A is a graph showing the ionomycin dose response, as measured byfluorescence emission of living cells that express β-lactamase reportergene, and which were contacted with a fluorogenic β-lactamase substrate.Because the NFAT response element usually requires both a calciumincrease and protein kinase C activation, these cells were also treatedwith 10 nm PMA FIG. 4B is a graph showing the PMA dose response ofliving cells that express a β-lactamase reporter gene, and which werecontacted with a fluorogenic β-lactamase substrate. In this case, allsamples were also treated with 2 μM ionomycin.

FIG. 5 is a graphic representation of the emission spectrum of theβ-lactamase substrate CCF2 before and after it is cleaved byβ-lactamase.

FIG. 6 shows the results of an NFAT β-lactamase transcription basedassay using a heterologouly express GPCR (Gq subtype) in the presence ofagonist, agonist and antagonist or solvent for agonist (“non-stimulated”control).

FIG. 7 shows activation of a Gαs subtype GPCR (panels A-C) and a Gαisubtype GPCR (panels D-F) using promiscuous Gα protein in a cell-based(transient transfection of all constructs) calcium indicator assay(FURA-PE3).

Panel A: 60 seconds after starting of the experiment, 10 μM agonistsolution was added to the cells transfected by pCIS/Gα 16 andGs-receptor expression plasmids.

Panel B: 60 seconds after starting of the experiment, 10 μM agoinstsolution was added to the cells transfected by both pCIS/Gα 16 alone.

Panel C: 60 seconds after starting of the experiment, 10 μM agonistsolution was added to the cells transfected by Gαs receptor expressionplasmid alone.

Panel D: 60 seconds after starting of the experiment, 10 μM agonistsolution was added to the cells transfected by pCIS/Gα16 andGαi-receptor expression plasmids.

Panel E: 60 seconds after starting of the experiment, 10 μM agonistsolution was added to the cells transfected by pCIS/Gα 16 alone.

Panel F: 60 seconds after starting of the experiment, 10 μM agonistsolution was added to the cells transfected by Gαi-receptor expressionplasmid alone.

FIG. 8 shows activation of a Gas subtype GPCR using promiscuous Gαprotein in a cell-based (stably transfected constructs for both thepromiscuous Gα-protein and GPCR) calcium indicator assay.

Panel A: Calcium imaging of the Gα15Gαs-receptor dual stable pool-2. 10μm agonist was added 40 seconds after the starting of the experiment.

Panel B: Calcium imaging of the Gαs-receptor stable pool-2. 10 μMagonist was added 40 seconds after the starting of the experiment.

Panel C: Clacium imaging of the Gα15 stable pool-H. 10 μM agoinst wasadded 40 seconds after the starting of the experiment.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein and the laboratory procedures in spectroscopy, drugdiscovery, cell culture, and molecular genetics, described below arethose well known and commonly employed in the art. Standard techniquesare typically used for preparation of signal detection, recombinantnucleic acid methods, polynucleotide synthesis, and microbial cultureand transformation (e.g., electroporation, and lipofection). Thetechniques and procedures are generally performed according toconventional methods in the art and various general references (seegenerally, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., and Lakowicz, J. R. Principles of Fluorescence Spectroscopy, NewYork: Plenum Press (1983) for fluorescence techniques, which areincorporated herein by reference) which are provided throughout thisdocument. Standard techniques are used for chemical syntheses, chemicalanalyses, and biological assays. As employed throughout the disclosure,the following terms, unless otherwise indicated, shall be understood tohave the following meanings:

“Fluorescent donor moiety” refers to the radical of a fluorogeniccompound, which can absorb energy and is capable of transferring theenergy to another fluorogenic molecule or part of a compound. Suitabledonor fluorogenic molecules include, but are not limited to, coumarinsand related dyes xanthene dyes such as fluoresceins, rhodols, andrhodamines, resorufins, cyanine dyes, bimanes, acridines, isoindoles,dansyl dyes, aminophthalic hydrazides such as luminol and isoluminolderivatives, aminophthalimides, aminonaphthalimides, aminobenzofurans,aminoquinolines, dicyanohydroquinones, and europium and terbiumcomplexes and related compounds.

“Quencher” refers to a chromophoric molecule or part of a compound,which is capable of reducing the emission from a fluorescent donor whenattached to the donor. Quenching may occur by any of several mechanismsincluding fluorescence resonance energy transfer, photoinduced electrontransfer, paramagnetic enhancement of intersystem crossing, Dexterexchange coupling, and excitation coupling such as the formation of darkcomplexes.

“Acceptor” refers to a quencher that operates via fluorescence resonanceenergy transfer. Many acceptors can re-emit the transferred as energy asfluorescence. Examples include coumarins and related fluorophores,xanthenes such as fluoresceins, rhodols, and rhodamines, resorufins,cyanines, difluoroboradiazaindacenes, and phthalocyanines. Otherchemical classes of acceptors generally do not re-emit the transferredenergy. Examples include indigos, benzoquinones, anthraquinones, azocompounds, nitro compounds, indoanilines, di- and triphenylmethanes.

“Binding pair” refers to two moieties (e.g. chemical or biochemical)that have an affinity for one another. Examples of binding pairs includeantigen/antibodies, lectin/avidin, target polynucleotide/probeoligonucleotide, antibody/anti-antibody, receptor/ligand, enzyme/ligandand the like. “One member of a binding pair” refers to one moiety of thepair, such as an antigen or ligand.

“Dye” refers to a molecule or part of a compound that absorbs specificfrequencies of light, including but not limited to ultraviolet light.The terms “dye” and “chromophore” are synonymous.

“Fluorophore” refers to a chromophore that fluoresces.

“Membrane-permeaiit derivative” refers a chemical derivative of acompound that has enhanced membrane permeability compared to anunderivativized compound. Examples include ester, ether and carbamatederivatives. These derivatives are made better able to cross cellmembranes, i.e. membrane permeant, because hydrophilic groups are maskedto provide more hydrophobic derivatives. Also, masking groups aredesigned to be cleaved from a precursor (e.g., fluorogenic substrateprecursor) within the cell to generate the derived substrateintracellularly. Because the substrate is more hydrophilic than themembrane permeant derivative it is now trapped within the cells.

“Isolated polynucleotide” refers a polynucleotide of genomic, cDNA, orsynthetic origin or some combination there of, which by virtue of itsorigin the “isolated polynucleotide” (1) is not associated with the cellin which the “isolated polynucleotide” is found in nature, or (2) isoperably linked to a polynucleotide which it is not linked to in nature.

“Isolated protein” refers a protein, usually of cDNA, recombinant RNA,or synthetic origin or some combination thereof, which by virtue of itsorigin the “isolated protein” (1) is not associated with proteins thatit is normally found with in nature, (2) is isolated from the cell inwhich it normally occurs, (3) is isolated free of other proteins fromthe same cellular source, e.g. free of human proteins, (4) is expressedby a cell from a different species, or (5) does not occur in nature.“Isolated naturally occurring protein” refers to a protein which byvirtue of its origin the “isolated naturally occurring protein” (1) isnot associated with proteins that it is normally found with in nature,or (2) is isolated from the cell in which it normally occurs or (3) isisolated free of other proteins from the same cellular source, e.g. freeof human proteins.

“Polypeptide” as used herein as a generic term to refer to nativeprotein, fragments, or analogs of a polypeptide sequence. Hence, nativeprotein, fragments, and analogs are species of the polypeptide genus.Preferred Gα polypeptides, include those with the polypeptide sequencerepresented in the SEQUENCE ID LISTING and any other protein havingactivity similar to such Gα proteins as measured by one or more of theassays described herein. SEQ. ID NO.: 1 is Gα₁₆ (murine). SEQ. ID NO. 2is Gα₁₅ (human). Gα polypeptides or proteins can include any proteinhaving sufficient activity for detection in the assays described herein.

“Naturally-occurring” as used herein, as applied to an object, refers tothe fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. A control sequence “operably linked” to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under conditions compatible with the control sequences, suchas when the appropriate molecules (e.g., inducers and polymerases) arebound to the control or regulatory sequence(s).

“Control sequence” refers to polynucleotide sequences which arenecessary to effect the expression of coding and non-coding sequences towhich they are ligated. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequence. The term“control sequences” is intended to include, at a minimum, componentswhose presence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

“Polynucleotide” refers to a polymeric form of nucleotides of at least10 bases in length, either ribonucleotides or deoxynucleotides or amodified form of either type of nucleotide. The term includes single anddouble stranded forms of DNA.

Two amino acid sequences are homologous if there is a partial orcomplete identity between their sequences. For example, 85% homologymeans that 85% of the amino acids are identical when the two sequencesare aligned for maximum matching. Gaps (in either of the two sequencesbeing matched) are allowed in maximizing matching; gap lengths of 5 orless are preferred with 2 or less being more preferred. Alternativelyand preferably, two protein sequences (or polypeptide sequences derivedfrom them of at least 30 amino acids in length) are homologous, as thisterm is used herein, if they have an alignment score of at more than 5(in standard deviation units) using the program ALIGN with the mutationdata matrix and a gap penalty of 6 or greater. See Dayhoff, M.O., inAtlas of Protein Sequence and Structure, 1972, Volume 5, NationalBiomedical Research Foundation, pp. 101-110, and Supplement 2 to thisvolume, pp. 1-10. The two sequences or parts thereof are more preferablyhomologous if their amino acids are greater than or equal to 30%identical when optimally aligned using the ALIGN program.

“Corresponds to” refers to a sequence that is homologous (i.e., isidentical, not strictly evolutionarily related) to all or a portion of areference sequence.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence,” “comparisonwindow,” “sequence identity,” “percentage of sequence identity” and“substantial identity.” A “reference sequence” is a defined sequenceused as a basis for a sequence comparison; a reference sequence may be asubset of a larger sequence, for example, as a segment of a full-lengthcDNA or gene sequence given in a sequence listing such as a SEQ. ID NO.1, or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 400 nucleotides in length, frequently atleast 600 nucleotides in length, and often at least 800 nucleotides inlength. Since two polynucleotides may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide sequence) that issimilar between the two polynucleotides, and (2) may further comprise asequence that is divergent between the two polynucleotides, sequencecomparisons between two (or more) polynucleotides are typicallyperformed by comparing sequences of the two polynucleotides over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window,” as used herein, refers to aconceptual segment of at least 20 contiguous nucleotide positionswherein a polynucleotide sequence may be compared to a referencesequence of at least 20 contiguous nucleotides and wherein the portionof the polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) of 20 percent or less as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. Optimal alignment ofsequences for aligning a comparison window may be conducted by the localhomology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482,by the homology alignment algorithm of Needleman and Wunsch (1970) J.Mol. Biol. 48: 443, by the search for similarity method of Pearson andLipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection, and the bestalignment (i.e., resulting in the highest percentage of homology overthe comparison window) generated by the various methods is selected..The term “sequence identity” means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence that has at least 50 percentsequence identity, preferably at least 60 to 70 percent sequenceidentity, more usually at least 80 percent sequence identity as comparedto a reference sequence over a comparison window of at least20nucleotide positions, frequently over a window of at least 25-50nucleotides, wherein the percentage of sequence identity is calculatedby comparing the reference sequence to the polynucleotide sequence whichmay include deletions or additions which total 20 percent or less of thereference sequence over the window of comparison.

As applied to proteins, the term “substantial identity” means that twoprotein sequences, when optimally aligned, such as by the programs GAPor BESTFIT using default gap weights, typically share at least 70percent sequence identity, preferably at least 80 percent sequenceidentity, more preferably at least 90 percent sequence identity, andmost preferably at least 95 percent sequence identity. Preferably,residue positions which are not identical differ by conservative aminoacid substitutions. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginne, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamic-aspartic, and asparagine-glutamine.

“Promiscuous Gα protein” refers to a protein with the promiscuouscoupling activity of one of the Gα proteins of the SEQ. ID listing.Preferably, the promiscuous Gα protein can couple to at least one GPCRthat normally couples to a Gα protein other than a promiscuous Gαprotein. Examples of Gα proteins, include Gαq, Gαs, Gαi and Gαl₁₂.Promiscuous Gα protein coupling activity can be measured with anendogenously or heterologously expressed GPCR using the assays describedherein. Preferably, a promiscuous Gα protein can couple to at least twodifferent types of GPCRs that normally couple to one of the following Gαproteins, Gαq, Gαs, Gαi and Gα12. More preferably, a promiscuous Gαprotein can couple to at least three different types of GPCRs thatnormally couple to one of the following Gα proteins, Gαq, Gαs, Gαi andGα12. Promiscuous Gα proteins permit coupling under conditions thatwould not occur with a Gα protein and a receptor of a different Gαsubtype, unless the Gα protein was expressed at sufficiently high levelsto promote coupling with a GFCR that is not its normal coupling partner.Examples of Gα₁₅ include (Wilke, T. M. et al., PNAS, Vol. 88 pp.10049-10053, 1991) and Gα₁₆ include (Amatruda, T. T. et al., PNAS, Vol.88 pp. 5587-5591, 1991). It is understood that promiscuous Gα proteinsdo not include members of Gαq, Gαs, Gαi and Gα12 proteins that couple toonly one type of GPCR

“Modulation” refers to the capacity to either enhance or inhibit afunctional property of biological activity or process (e.g., enzymeactivity or receptor binding); such enhancement or inhibition may becontingent on the occurrence of a specific event, such as activation ofa signal transduction pathway, and/or may be manifest only in particularcell types.

The term “modulator” refers to a chemical compound (naturally occurringor non-naturally occurring), such as a biological macromolecule (e.g.,nucleic acid, protein, non-peptide, or organic molecule), or an extractmade from biological materials such as bacteria, plants, fungi, oranimal (particularly mammalian) cells or tissues. Modulators areevaluated for potential activity as inhibitors or activators (directlyor indirectly) of a biological process or processes (e.g., agonist,partial antagonist, partial agonist, inverse agonist, antagonist,antineoplastic agents, cytotoxic agents, inhibitors of neoplastictransformation or cell proliferation, cell proliferation-promotingagents, and the like) by inclusion in screening assays described herein.The activity of a modulator may be known, unknown or partially known.

“Sequence homology” refers to the proportion of base matches between twonucleic acid sequences or the proportion amino acid matches between twoamino acid sequences. When sequence homology is expressed as apercentage, e.g., 50%, the percentage denotes the proportion of matchesover the length of sequence from a desired sequence (e.g., SEQ. IDNO. 1) that is compared to some other sequence. Gaps (in either of thetwo sequences) are permitted to maximize matching; gap lengths of 15bases or less are usually used, 6 bases or less are preferred with 2bases or less more preferred.

The term “test chemical” refers to a chemical to be tested by one ormore screening method(s) of the invention as a putative modulator.

The terms “label” or “labeled” refers to incorporation of a detectablemarker, e.g., by incorporation of a radio labeled amino acid orattachment to a polypeptide of biotinyl moieties that can be detected bymarked avidin (e.g., streptavidin containing a fluorescent marker orenzymatic activity that can be detected by optical, or colorimetricmethods). Various methods of labeling polypeptides and glycoproteins areknown in the art and may be used. Examples of labels for polypeptidesinclude, but are not limited to, the following: radioisotopes (e.g., ³H,¹⁴C, ³⁵S, ¹²⁵I, ¹³¹I, fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (or reporter genes) (e.g.,horseradish peroxidase, β-galactosidase, β-latamase, luciferase,alkaline phosphatase), chemiluminescent, biotinyl groups, predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, epitope tags). In some embodiments, labels are attachedby spacer arms of various lengths to reduce potential steric hindrance.

“Fluorescent label” refers to incorporation of a detectable marker,e.g., by incorporation of a fluorescent moiety to a chemical entity thatbinds to a target or attachment to a polypeptide of biotinyl moietiesthat can be detected by avidin (e.g., streptavidin containing afluorescent label or enzymatic activity that can be detected byfluorescence detection methods). Various methods of labelingpolypeptides and glycoproteins are known in the art and may be used.Examples of labels for polypeptides include, but are not limited to dyes(e.g., FITC and rhodamine), intrinsically fluorescent proteins, andlanthanide phosphors. In some embodiments, labels are attached by spacerarms of various lengths to reduce potential steric hindrance.

“Reporter gene” refers to a nucleotide sequence encoding a protein thatis readily detectable either by its presence or activity, including, butnot limited to, luciferase, green fluorescent protein, chloramphenicolacetyl transferase, β-galactosidase, secreted placental alkalinephosphatase, β-lactamase, human growth hormone, and other secretedenzyme reporters. Generally, reporter genes encode a polypeptide nototherwise produced by the host cell, which is detectable by analysis ofthe cell(s), e.g., by the direct fluorometric, radioisotopic orspectrophotometric analysis of the cell(s) and preferably without theneed to kill the cells for signal analysis. Preferably, the gene encodesan enzyme, which produces a change in fluorometric properties of thehost cell, which is detectable by qualitative, quantitative orsemi-quantitative function of transcriptional activation. Exemplaryenzymes include esterases, phosphatases, proteases (tissue plasminogenactivator or urokinase) and other enzymes whose function can be detectedby appropriate chromogenic or fluorogenic substrates known to thoseskilled in the art.

“Signal transduction detection system” refers to system for detectingsignal transduction across a cell membrane, typically a cell plasmamembrane. Such systems typically detect at least one activity orphysical property directly or indirectly associated with signaltransduction. For example, an activity or physical property directlyassociated with signal transduction is the activity or physical propertyof either the receptor (e.g., GPCR),or a coupling protein (e.g., a Gαprotein). Signal transduction detection systems for monitoring anactivity or physical property directly associated with signaltransduction, include GTPase activity, and conformational changes. Anactivity or physical property indirectly associated with signaltransduction is the activity or physical property produced by a moleculeother than by either the receptor (e.g., GPCR), or a coupling protein(e.g., a Gα protein) and associated with receptor (e.g., GPCR), or acoupling protein (e.g., a Gα protein). Such indirect activities andproperties include changes in intracellular levels of molecules (e.g.,ions (e.g., Ca, Na or K), second messenger levels (e.g., cAMP, cGMP andinostol phosphate)), kinase acitvites, transcriptional activity,enzymatic activity, phospholipase activities, ion channel activities andphosphatase activites. Signal transduction detection systems formonitoring an activity or physical property indirectly associated withsignal transduction, include transcriptional-based assays, enzymaticassays, intracellular ion assays and second messenger assays.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(ed. Parker, S., 1985), McGraw-Hill, San Francisco, incorporated hereinby reference).

A DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention provides cells and methods for screening or characterizingG-protein coupled receptors (GPCRs), ligands for GPCRs, and compoundsthat modulate signal transduction (e.g., agonists and antagonists). Theterm “G-protein coupled receptor” is used herein in accordance with itsconventional definition. Such receptors are cell surface receptors thattypically contain seven transmembrane regions and that transduce signals(e.g., sensory, hormonal, and neurotransmitter signals) fromextracellular environments to intracellular environments.

Included within the invention are cells that are useful for expressing Gproteins and practicing methods of the invention. A preferred cell is astable, isolated cell that comprises a promiscuous Gα protein constructcomprising a promoter operably linked to a gene (or polynucleotide) thatencodes a polypeptide with the biological activity of a promiscuous Gαprotein. A “stable isolated cell” of the invention is a cell thatretains a construct typically longer than at least 3 to 4 passages intissue culture, preferably longer than 6 to 10 passages and mostpreferably longer than about 12 passages. An “isolated” cell refers to acell in an in vitro state (e.g., a cell of a mammalian tissue culture).The cells that are useful in the invention include both eukaryotic andprokaryotic cells that contain the constructs described herein.Preferably, the cell is a cell of a mammalian cell line (e.g., a COS-7cell); human cells are also preferred (e.g., a human T lymphocyte).Although not preferred, yeast cells can also be used.

A “construct,” when used in the context of molecular biology, is anygenetically engineered nucleic acid (e.g., a plasmid, restrictionfragment or an engineered chromosome). As used herein, a “promoter” isthe minimal sequence sufficient to direct transcription of a gene(including a cDNA encoding a protein) in an eukaryote. Preferably, thepromoter is derived from an eukaryotic gene or a virus that can directtranscription in an eukaryotic cell. A promoter can include a TATA box,a CAAT box, and a transcriptional start site. The term “gene” refers toa polynucleotide that encodes a protein, such as a cDNA encoding aprotein.

Polypeptides that have the biological activity of a Gα protein are thosepolypeptides that are able to transduce a signal (includingextracellular signals) to an effector(s) in a G-protein signalingpathway. Typically, such a polypeptide or protein, in its inactivestate, is associated with GDP and the βγ dimer of a G-protein. In its“active” state, the polypeptide typically is associated with GTP anddisassociated from the βγ dimer of a G-protein. The unassociated Gαprotein is able to transduce a signal to an effector in the G-proteinsignaling pathway. Examples promiscuous Gα proteins include promiscuousGα₁₆ protein and a promiscuous Gα₁₅ protein. Either promiscuous Gα₁₆protein or a promiscuous Gα₁₅ protein can couple to a GPCR that normallycouples to G_(i), G_(s)or G_(q) (see FIG. 1). Preferably, thepromiscuous Gα protein employed in the invention has the ability tocouple with specificity to an effector in the G-protein signalingpathway. For example, the promiscuous Gα₁₆ and Gα₁₅ proteins each retainthe ability to specifically activate the β isoform of phospholipase C.

Preferably, the nucleotide sequence of a promiscuous Gα protein has atleast 70% (more preferably, at least 80% or 95%) sequence identity tothe nucleotide sequence of Gα₁₆ (SEQ ID NO: 1) and/or Gα₁₅ (SEQ ID NO:2). Other preferred promiscuous Gα proteins are those that are encodedby degenerate variants of the nucleotide sequences of promiscuous Gα₁₆(SEQ ID NO: 1) and/or Gα₁₅ (SEQ ID NO: 2). A “degenerate variant” of anucleotide sequence is a nucleotide sequence that encodes the same aminoacid sequence as a given nucleotide sequence, but in which at least onecodon in the nucleotide sequence is different, because two or moredifferent codons can encode the same amino acid. Accordingly, numerousdegenerate variants can encode the promiscuous Gα proteins of SEQ ID NO:3 and SEQ ID NO: 4. Other preferred promiscuous Gα protein are thosethat are encoded by conservative variations of the nucleotide sequencesof Gα₁₆ (SEQ ID NO: 1) and/or Gα₁₅ (SEQ ID NO: 2). A “conservativevariation” denotes the replacement of an arnino acid residue by another,biologically similar, residue. Examples of conservative variationsinclude the substitution of one hydrophobic residue, such as isoleucine,valine, leucine, or methionine, for another, or the substitution of onepolar residue for similar polar residue, such as the substitution ofarginine for lysine, glutamic acid for aspartic acid, or glutamine forasparagine, and the like.

In some embodiments of the invention it will be desirable to control thelevel of promiscuous Gα protein expression. High levels of promiscuousGα protein in a cell can deleteriously alter cell metabolism that canresult in cell instability. High levels of promiscuous Gα protein in acell (or normal Gα protein) can also produce high basal activities GPCRsthat results in high background activities, which is not desirable formethods described herein, such as screening chemicals that may modulatereceptor activity. Typically, cells having endogenously low levels ofnormal Gα protein are used. Basal activity levels of GPCRs can be easilytested in a potential cell type to be used for screening with a signaltransduction detection system to detect the affect of endogenouslyexpressed G-proteins. Basal activity levels of GPCRs can also be easilytested with either endogenously expressed or heterologously expressedGPCRs in cells expressing a promiscuous Gα protein or other G-proteins.

With GPCRs normally having high basal activity, controlled levels ofpromiscuous Gα protein can help reduce background activity in a cellwhile achieving suitable coupling for testing putative modulators of areceptor. The amount promiscuous Gα protein expressed in a cell can betitrated by using, or selecting for, either a weak promoter or aninducible promoter. An inducible promoter offers the advantage, comparedto a weak promoter, of regulatable expression of promiscuous Gα protein.By using an inducible promoter the amount inducer can be used tooptimize the signal to noise ratio of a screen for GPCR modulators byadjusting the amount of promiscuous Gα protein expression the cell.

An “inducible” promoter is a promoter that, in the absence of an inducer(e.g., doxycyclin) does not direct expression, or directs low levels ofexpression (e.g., produces less than 500 proteins per cell at steadystate) of an operably linked gene (including CDNA). In the presence ofan inducer, expression promiscuous Gα protein directed by the induciblepromoter is typically increased at least 3-fold (preferably at least10-100- or 1,000-fold). Other useful inducible promoters include thosethat are inducible by IPTG or ecdysone. If desired, an induciblepromoter can include a first promoter (e.g., a cytomegalovirus promoter)operably linked to a tet operator to regulate the first promoter (see,Gossen and Bujard, 1992, Proc. Natl. Acad. Sci. 89:5547-5551).

Many embodiments of the invention will include a polynculeotide encodinga GPCR not naturally occurring in the cell and a promiscuous Gα proteinconstruct. The GPCR will typically not be under the control of thecontrol sequence controlling promiscuous Gα protein expression. The GPCRmaybe a GPCR of known function or of protein of unknown function, suchas an orphan GPCR. Promoters known in the art can be used to eitherconstitutively or inducible express the receptor or putative receptor.

If desired, a cell of the invention can contain a polynucleotide havinga control sequence and encoding a protein useful in signal transductiondetection system. The construct is designed to detect activation of a Gαprotein. This second construct is typically located on a second vector.It can include a reporter gene that is operably linked to a promoterthat is modulated (directly or indirectly) by an active promiscuous Gαprotein. Preferably, the expression of the reporter gene can be detectedby detecting a change in fluorescence emission of a sample that containsthe cell.

For instance, the reporter system described in PCT publicationWO96/30540 (Tsien) has significant advantages over existing reportersfor gene integration analysis, as it allows sensitive detection andisolation of both expressing and non-expressing single living cells.This assay system uses a non-toxic, non-polar fluorescent substrate,which is easily loaded and then trapped intracellularly. Cleavage of thefluorescent substrate by β-lactamase lactamase yields a fluorescentemission shift as substrate is converted to product. Because theβ-lactamase reporter readout can be ratiometric, it is unique amongreporter gene assays in that it controls variables such as the amount ofsubstrate loaded into individual cells. The stable, easily detected,intracellular readout simplifies assay procedures by eliminating theneed for washing steps, which facilitates screening with cells using theinvention. Preferably, a ratiometric fluorescent signal transductiondetection system can be used with the invention. Preferred fluorogenicsubstrates are described in the Examples.

Other reporter genes such as polynucleotides encoding a polypeptidehaving the biological activity of green fluorescent protein (GFP) can beused.

A promoter is considered to be “modulated” by an active, promiscuous Gαprotein when the expression of a reporter gene to which the promoter isoperably linked is either increased or decreased upon activation of thepromiscuous Gα protein. It is not necessary that the active, promiscuousGα protein directly modulate reporter gene expression.

For example, embodiments of the invention presume that activation ofGα₁₅ or Gα₁₆ can, through a G-protein signaling pathway, activate PLCβ,which in turn increases intracellular calcium levels. An increase incalcium levels can lead to modulation of a “calcium-responsive” promoterthat is part of a signal transduction detection system, i.e., a promoterthat is activated (e.g., a NFAT promoter) or inhibited by a change incalcium levels. One example of an NFAT-DNA binding site is found inShaw, et al. Science 291:202-205 1988. Likewise, a promoter that isresponsive to changes in protein kinase C levels (i.e., a “proteinkinase C-responsive promoter”) can be modulated by an active Gα proteinthrough G-protein signaling pathway. The cells described above can alsoinclude a G-protein coupled receptor. Genes encoding numerous GPCRs havebeen cloned (Simon et al., 1991, Science 252:802-808), and conventionalmolecular biology techniques can be used to express a GPCR on thesurface of a cell of the invention. Preferably, the sum responsivepromoter allows only a relatively short lag (e.g., less than 90 minutes)between engagement of the GPCR and transcriptional activation. Apreferred responsive promoter includes the nuclear factor of activatedT-cell promoter (Flanagan et al., 1991, Nature 352:803-807).

Many cells can be used in the invention, particularly for heterologousexpression of a GPCR. Such cells include, but are not limited to; babyhamster kidney (BHK) cells (ATCC No. CCL10), mouse L cells (ATCC No.CCLI.3), Jurkats (ATCC No. TIB 152) and 153 DG44 cells [see, Chasin(1986) Cell. Molec. Genet. 12: 555] human embryonic kidney (HEK) cells(ATCC No. CRL1573), Chinese hamster ovary (CHO) cells (ATCC Nos.CRL9618, CCL61, CRL9096), PC12 cells (ATCC No. CRL17.21) and COS-7 cells(ATCC No. CRL1 651). Preferred cells for heterologous cell surfaceprotein expression are those that can be readily and efficientlytransfected. Preferred cells include Jurkat cells CHO cells and HEK 293cells, such as those described in U.S. Pat. No. 5,024,939 and byStillman et al. (1 985) Mol. Cell. Biol. 5: 2051-2060.

GPCRs that can be used with the invention include, but are not limitedto, muscarinic receptors, e.g., human M2 (GenBank accession #M16404);rat M3 (GenBank accession #M16407); human M4 (GenBank accession#M16405); human M5 (Bonner, et al., (1988) Neuron 1, pp. 403-410); andthe like; neuronal nicotinic acetylcholine receptors, e.g., the humanα₂, α₃, and β₂, subtypes disclosed in U.S. Ser. No. 504,455 (filed Apr.3, 1990, which is hereby expressly incorporated by reference 0herein inits entirety); the human α₅ subtype (Chini, et al. (1992) Proc. Natl.Acad. Sci. U.S.A. 89: 1572-1576), the rat α₂ subunit (Wada, et al.(1988) Science 240, pp. 330-334); the rat α₃ subunit (Boulter, et al.(1986) Nature 319, pp. 368-374); the rat α₄ subunit (Goldman, et al.(1987) Cell 48, pp. 965-973); the rat α₅ subunit (Boulter, et al. (1990)I. Biol. Chem. 265, pp. 4472-4482); the chicken α₇ subunit (Couturieret. al. (1990) Neuron 5: 847-856); the rat β₂ subunit (Deneris, et al.(1988) Neuron 1, pp. 45-54) the rat β₃ subunit (Deneris, et al. (1989)J. Biol. Chem. 264, pp. 6268-6272); the rat β₄ subunit (Duvoisin, et al.(1989) Neuron 3, pp. 487-496); combinations of the rat α subunits, and sβ subunits and a and p subunits; GABA receptors, e.g., the bovine x, andβ₁, subunits (Schofield, et al. (1987) Nature 328, pp.. 221-227); thebovine X₂, and X₃, subunits (Levitan, et al. (1988) Nature 335, pp.76-79); the γ-subunit (Pritchett, et al. (1989) Nature 338, pp.582-585); the β₂, and β₃, subunits (Ymer, et al. (1989) EMBO J. 8, pp.1665-1670); the 8 subunit (Shivers, B. D. (1989) Neuron 3, pp. 327-337);and the like; glutamate receptors, e.g., rat GluR1 receptor (Hollman, etal. (1989) Nature 342, pp. 643-648); rat GluR2 and GluR3 receptors(Boulter et al. (1990) Science 249:1033-1037; rat GluR4 receptor(Keinanen et al. (1990) Science 249: 556-560); rat GluR5 receptor(Bettler et al. (1990) Neuron 5: 583-595); rat GluR6 receptor (Egebjerget al. (1991) Nature 351: 745-748); rat GluR7 receptor (Bettler et al.(1992) neuron 8:257-265); rat NMDAR1 receptor (Moriyoshi et al. (1991)Nature 354:31-37 and Sugihara et al. (1992) Biochem. Biophys. Res. Comm.185:826-832); mouse NMDA el receptor (Meguro et al. (1992) Nature 357:70-74); rat NMDAR2A, NMDAR2B and NMDAR2C receptors (Monyer et al. (1992)Science 256: 1217-1221); rat metabotropic mGluR1 receptor (Houamed etal. (1991) Science 252: 1318-1321); rat metabotropic mGluR2, mGluR3 andmGluR4 receptors (Tanabe et al. (1992) Neuron 8:169-179); ratmetabotropic mGluR5 receptor (Abe et al. (1992) I. Biol. Chem. 267:13361-13368); and the like; adrenergic receptors, e.g., human β1(Frielle, et al. (1987) Proc. Natl. Acad. Sci. 84, pp. 7920-7924); humanα₂ (Kobilka, et al. (1987) Science 238, pp. 650-656); hamster β₂ (Dixon,et al. (1986) Nature 321, pp. 75-79); and the like; dopamine receptors,e.g., human D2 (Stormann, et al. (1990) Molec. Pharm. 37, pp. 1-6);mammalian dopamine D2 receptor (U.S. Pat. No. 5,128,254); rat (Bunzow,et al. (1988) Nature 336, pp. 783-787); and the like; and the like;serotonin receptors, e.g., human 5HT1a (Kobilka, et al. (1987) Nature329, pp. 75-79); serotonin 5HT1C receptor (U.S. Pat. No. 4,985,352);human 5HT1D (U.S. Pat. No. 5,155,218); rat 5HT2 (Julius, et al. (1990)PNAS 87, pp.928-932); rat 5HT1c (Julius, et al. (1988) Science 241, pp.558-564), and the like.

If desired (e.g., for commercial purposes), a cell(s) of the inventioncan packaged into a container that is packaged within a kit. Such a kitmay also contain any of the various isolated nucleic acids, antibodies,proteins, signal transduction detection systems, substrates, and/ordrugs described herein, known in the art or developed in the future. Atypical kit also includes a set of instructions for any or all of themethods described herein.

METHODS OF THE INVENTION

The invention provides several methods for cloning or characterizingGPCRs, screening or characterizing ligands (e.g., known ligands) ofGPCRs, and identifying or characterizing compounds that modulate signaltransduction. For example, the invention provides a method fordetermining whether a “target” polypeptide is a GPCR for a given ligand.The method involves expressing a target polypeptide in a cell describedherein that comprises a reporter gene construct (e.g., a constructencoding β-lactamase reporter gene operably linked to a NFAT promoter).In this method, the test polypeptide is contacted with a chosen ligand,usually of established activity, and a change in reporter geneexpression is detected. A “target” polypeptide, which is usually a GPCR,is any polypeptide expressed by a cell that can be assayed for activityusing the present invention.

Similar methods can be used to test ligands and compounds using GPCRs ofknown, partially known and unknown function. A test ligand is a moleculethat can be assayed for its ability to bind to a GPCR. A test compoundis a molecule that can be assayed for its ability to modulator of signaltransduction. Often, such a target polypeptide, test ligand, or testcompound is, because of its sequence or structure, suspected of beingable to function in a given capacity. Nonetheless, randomly chosentarget polypeptides, test ligands, and test compounds also can be usedin the methods described herein, and with techniques known in the art ordeveloped in the future. For example, expression of target polypeptidesfrom nucleic acid libraries, can be used to identify proteins involvedin signal transduction, such as orphan GPCRs. For instance, thistechnique can be used to identify physiologically responsive receptors(e.g., taste-responsive GPCRs) where the ligand responsible for inducinga physiological event is known (e.g., a given taste sensation is known).

The invention also includes enhancement of reporter gene expression in asignal transduction detection system. This particularly useful forimproving the signal to noise ratio in a screening assay. It generallyinvolves contacting the cell with a molecule (“subthreshold regulatingmolecule”) that alters the activity of a cellular process to a levelsubthreshold to the activation of a cellularly responsive controlsequence that is operably linked to the reporter gene. Because the levelof cellular activity is subthreshold, the reporter gene has a lowexpression level. The reporter gene system, however, is poised foractivation by a change in cellular process induced by either a testchemical, test ligand or expression of target protein. Such cellularlyresponsive control sequences can be responsive elements known in the artin other applications. Such response elements, however, do not need beresponsive to their naturally occurring signal, since the assay mayoccur in cells lacking the required constituents for activation by anaturally occurring signal. The subthreshold regulating molecule caneither increase or decrease the activity of the cellular process. It isunderstood that the cellular process may not only be “classic” cellularprocess, such as an enzymatic activity, but it also includes levels ofcellular entities (e.g., ions, metabolites and second messengers) orother measurable properties of the cell (e.g., cell volume, chromatindensity, etc.). Cells described herein are preferred for this method.Other cells, however, can be used as well which express Gα proteinsendogenously, or heterologously.

For example, in order to enhance detection of expression of a reportergene, the cell can be contacted with a compound (e.g., a calciumionophore) that increases calcium levels inside of the cell. Byincreasing calcium levels inside the cell, the probability thatactivation of a G-protein will activate expression of a reporter gene isgreatly enhanced. Preferably, the calcium levels are increased to alevel that is just below the threshold level for activation of acalcium-responsive promoter, such as an NFAT promoter (see FIG. 2). Inpractice, ionomycin typically is added at a concentration of about 0.01to 3 μM, preferably 0.03 μM. Cells described herein are preferred forthis method. Other cells, however, can be used as well which express Gαproteins endogenously, or heterologously.

In an alternate method of enhancing a signal transduction detectionsystem, thapsigargin is added to the cell to set intracellular calciumlevels at subthreshold levels to enhance reporter gene activation.Thapsigargin is added to the cell at a concentration of about 1 to 50nM, with the effect of partially depleting intracellular calcium poolsand slowing the re-filling of such pools (Thastrup et al., 1990, Proc.Natl. Acad. Sci. 87:2466-2470). If desired, thapsigargin can be used ata higher concentration (e.g., 200 nM to 1 μM) in a “Ca⁺²-clamp”protocol, in which membrane potential is used to set the baselinecalcium concentration (Negulescu et al., 1994, Proc. Natl. Acad. Sci.91:2873-2877). This can be applied to screening for modulators of signaltransduction using a reporter gene system with a calcium-responsivepromoter. Cells described herein are preferred for this method. Othercells, however, can be used as well which express Gα proteinsendogenously, or heterologously.

In yet another method of the invention, conventional molecular biologytechniques can be used to express a calcium modulating ligand in cells,and thereby increase calcium levels (Bram et al., 1994, Nature371:355-358). This can be applied to screening for modulators of signaltransduction using a reporter gene system with a calcium-responsivepromoter. Cells described herein are preferred for this method. Othercells, however, can be used as well which express Gα proteinsendogenously, or heterologously.

A related method of the invention for enhancing detection of expressionof the reporter gene involves contacting the cell with an activator ofprotein kinase C. Typically, this method involves contacting the cellwith 1 to 3 nM of phorbol myristate acetate (PMA) or another phorbolester, preferably PMA is used at a concentration of 0.3 nM. The PMAconcentration can be titrated to achieve sub threshold levels. Variousanalogs of PMA that retain this activity are known in the art, and canbe used in the invention. Cells described herein are preferred for thismethod. Other cells, however, can be used as well which express Gαproteins endogenously, or heterologously.

The invention also provides a method for determining whether a “test”ligand is a ligand for a given GPCR. In this method, a selected GPCR isexpressed in a cell, such as a cell of the invention, which contains aconstruct and encodes a reporter gene. The cell is contacted with a testligand, and a change in expression of the reporter gene is detected.This method is particularly well suited for identifying a ligand notknown to bind to the receptor and it can also be used to determinereceptor selectivity. In this method, the change in expression of thereporter gene can be compared for a sample of cells in the presence,versus in the absence, of the test ligand in order to identify ligandspecific activation. Cells described herein are preferred for thismethod. Other cells, however, can be used as well which express Gαproteins endogenously, or heterologously..

The aforementioned methods can readily be adapted to provide a methodfor characterizing the ability of a ligand to interact with a panel ofGPCRs of interest. In such an assay, the first GPCR of interest isexpressed in a cell, such as a cell of the invention, that contains aconstruct encoding a reporter gene. In a second cell (in a second,separate sample), a second GPCR of interest is expressed along withreporter gene system. Additional GPCRs can be expressed in additionalcells with reporter gene systems. Typically, these cells differ onlywith respect to the GPCR that is expressed. Each sample of cells iscontacted with the “test” ligand of interest, and a change in reportergene expression is detected for each cell sample. By comparing thechanges in expression of the reporter gene between cell samples, one cancharacterize the functional activity of the ligand. This method isparticularly well suited for assaying the ability of a known ligand tointeract with several GPCRs that are known to be related. Thus theselectivity of the ligand can be determined. For example, variousmuscarinic receptors (e.g., M₁, M₂, and M₃) can be expressed,separately, on a cell. If desired, various modulators of G-proteinactivity (e.g., agonists and antagonists) can be characterized in avariation of this method. Cells described herein are preferred for thismethod. Other cells, however, can be used as well which express Gαproteins endogenously, or heterologously.

The invention also provides a general method for determining whether atest compound modulates signal transduction in a cell. This method alsoemploys a cell, such as a cell of the invention, that includes aconstruct, and that expresses a reporter gene. In this method, the cellexpresses a GPCR, and the cell is contacted with a ligand that, in theabsence of a test compound, activates signal transduction. The cell isalso contacted with a test compound, and a change in expression of thereporter gene indicates that the test compound modulates signaltransduction in the cell.

In a variation of this method, the invention provides a “receptor-less”method for determining whether a test compound modulates signaltransduction. In this variation, the cell is not engineered to express aGPCR. In lieu of contacting the cell with a ligand, the cell iscontacted with a compound that directly activates a Gα protein encodedby a construct within the cell. Examples of such compounds includemastoparan (Calbiochem) and aluminum fluoride. These compounds typicallyare used at concentrations of 0.5 to 5 mM. A change in expression of areporter gene indicates that the test compound modulates signaltransduction in the cell. Such a change also indicates that the compoundaffects signaling events that occur subsequent to receptor signaling inthe signaling pathway.

The invention also provides a method for determining whether a testpolypeptide is a GPCR for a given ligand, without employing a secondgenetic construct expressing a reporter gene. In this method, a testpolypeptide is expressed in a stable, isolated cell that carries agenetic construct that includes a promoter operably linked to a genethat encodes a polypeptide having the biological activity of apromiscuous Gα protein. The test polypeptide is contacted with a ligand,and an increase in calcium levels within the cell is detected. Any ofthe art-known methods for detecting a change in calcium levels can beused in this method (Negulescu and Machen, 1990, Meth. in Enzymol.192:38-81). In a preferred method, the increase is detected bycontacting the cell with fura-2 (available from Molecular Probes;Eugene, Oreg.) and detecting a change in fluorescence emission of asample that includes the cell.

The invention offers several advantages. By employing promiscuousG-proteins, the invention allows the use of a single intracellularsignaling pathway (e.g., activation of PLCβ) to analyze GPCRs thatnormally couple specifically to G-proteins of a single family. Byproviding methods that employ living cells, the invention allows areceptor or ligand that is identified in an assay to be cloned. Byemploying fluorescent detection methods, the invention, in variousembodiments, allows a practitioner to characterize a single cell.Accordingly, convenient cell-sorting methods, such as FACS, can be usedto analyze and isolate cells. The fluorescent assays employed in theinvention also provide a stable, non-labile indicator of G-proteinactivation. Such a stable signal (lasting up to twelve hours) allows apractitioner to analyze numerous samples in parallel, thus rendering theinvention useful for high throughput screening of “test” polypeptides,ligands, and compounds. The invention provides, for the first time, anassay for associating occupancy of any GPCR with gene expression, asdetected by a fluorescence emission. In addition, by providing methodsfor enhancing detection of G-protein activation, the invention providesa sensitive assay for detecting low levels, or brief activation, of aG-protein.

The kits can be produced to accomplish the methods described herein.Such kits can include the polynucleotides for GPCR expression, cells forGPCR expression or Gα protein expression and signal transductiondetection systems, such reporter gene systems.

EXAMPLES

The following examples are intended to illustrate but not limit theinvention. While they are typical of those methods that might be used,other procedures known to those skilled in the are may alternatively beused.

Example 1 Synthesis of a β Lactamase Substrate (Compound 7b)

For synthesis of 2,4 dihydroxy-5-chlorobenzaldehyde, 21.7 g (0.15 Mol)4-chlororesorcinol were dissolved in 150 ml dry diethyl ether and 27 gfinely powdered zinc (II) cyanide and 0.5 g potassium chloride wereadded with stirring. The suspension was cooled on ice. A strong streamof hydrogen chloride gas was blown into the solution with vigorousstirring. After approximately 30 minutes the reactants were dissolved.The addition of hydrogen chloride gas was continued until it stoppedbeing absorbed in the ether solution (approx. 1 hour). During this timea precipitate formed. The suspension was stirred for one additional houron ice. Then the solid was let to settle. The ethereal solution waspoured from the solid. The solid was treated with 100 g of ice andheated to 100° C. in a water bath. Upon cooling the product crystallizedin shiny plates from the solution. They were removed by filtration ondried over potassium hydroxide. The yield was 15.9 g (0.092 Mol, 61%).¹H NM (CDCl₃): δ 6.23 ppm (s, 1H, phenol), δ 6.62 ppm (s, 1H, phenyl), δ7.52 ppm (s, 1H, phenyl), δ 9.69 ppm (s, 1H, formyl), δ 11.25 ppm (s,1H, phenol).

To prepare 3-carboxy 6-chloro 7-hydroxy coumarin, 5.76 g (0.033 Mol)2,4-dihydroxy-5-chlorobenzaldehyde and 7.2 g (0.069 Mol) malonic acidwere dissolved in 5 ml warm pydine. 75 μl Aniline were stirred into thesolution and the reaction let to stand at room temperature for 3 days.The yellow solid that formed was broken into smaller pieces and 50 mlethanol was added. The creamy suspension was filtered through a glassfrit and the solid was washed three times with 1 N hydrochloric acid andthen with water. Then the solid was stirred with 100 ml ethyl acetate,150 ml ethanol and 10 ml half concentrated hydrochloric acid. Thesolvent volume was reduced in vacuo and the precipitate recovered byfiltration, washed with diethyl ether and dried over phosphorouspentoxide. 4.97 g (0.021 Mol, 63%) of product was obtained as a whitepowder. ¹H NMR (dDMSO): δ 6.95 ppm (s, 1H), δ 8.02 ppm (s, 1H), δ 8.67ppm (s, 1H).

To prepare 7-butyryloxy-3-carboxy-6-chlorocoumarin, 3.1 g (12.9 mMol)3-carboxy-6-chloro-7-hydroxycoumarin were dissolved in 100 ml dioxaneand treated with 5 ml butyric anhydride, 8 ml pyridine and 20 mgdimethyl aminopyridine at room temperature for two hours. The reactionsolution was added with stirring to 300 ml heptane upon which a whiteprecipitate formed. It was recovered by filtration and dissolved in 150ml ethyl acetate. Undissolved material was removed by filtration and thefiltrate extracted twice with 50 ml 1 N hydrochloric acid/brine (1:1)and then brine. The solution was dried over anhydrous sodium sulfate.Evaporation in vacuo yielded 2.63 g (8.47 mMol, 66%) of product. ¹H NMR(CDCl₃): δ 1.08 ppm (t, 3H, J=7.4 Hz, butyric methyl), δ 1.85 ppm (m,2H, J₁ δ J₂=7.4 Hz, butyric methylene), δ 2.68 ppm (t, 2H, J=7.4 Hz,butyric methylene), δ 7.37 ppm (s, 1H, coumarin), δ 7.84 ppm (s, 1H,coumarin), δ 8.86 ppm (s, 1H, coumarin).

Preparation of7-butyryloxy-3-benzyloxycarbonylmethylaminocarbonyl-6-chlorocoumarin iseffected as follows. 2.5 g (8.06 mMol)7-Butyryloxy-3-carboxy-6-chlorocoumarin, 2.36 g hydroxybenztriazolehydrate (16 mMol) and 1.67 g (8.1 mMol) dicyclohexyl carbodiimide weredissolved in 30 ml dioxane. A toluene solution of O-benxylglycine[prepared by extraction of 3.4 g (10 mMol) benzylglycine tosyl salt withethyl acetate-toluene-saturated aqueous bicarbonate-water (1:1:1:1, 250ml), drying of the organic phase with anhydrous sodium sulfate andreduction of the solvent volume to 5 ml] was added drop wise to thecoumarin solution. The reaction was kept at room temperature for 20hours after which the precipitate was removed by filtration and washedextensively with ethylacetate and acetone. The combined solventfractions were reduced to 50 ml on the rotatory evaporator upon whichone volume of toluene was added and the volume further reduced to 30 ml.The precipitating product was recovered by filtration and dissolved in200 ml chloroform-absolute ethanol (1:1). The solution was reduced to 50ml on the rotatory evaporator and the product filtered off and dried invacuo yielding 1.29 g of the title product. Further reduction of thesolvent volume yielded a second crop (0.64 g). Total yield: 1.93 g (4.22mMol, 52%). ¹H NMR (CDCl₃): δ 1.08 ppm (t, 3H, J=7.4 Hz, butyricmethyl), 1.84 ppm (m, 2H, J₁ δ J₂=7.4 Hz, butyric methylene), δ 2.66 ppm(t, 2H, J 7.4 Hz, butyric methylene), δ 4.29 ppm (d, 2H, J 5.5 Hz,glycine methylene), δ 5.24 ppm (s, 2H, benzyl), δ 7.36 ppm (s, 1H,coumarin), δ 7.38 ppm (s, 5H, phenyl), δ 7.77 ppm (s, 1H, coumarin), δ8.83 ppm (s, 1H, coumarin), δ 9.15 ppm (t, 1H, J 5.5 Hz, amide).

7-Butyryloxy-3-carboxymethylaminocarbonyl-6-chlorocoumarin was preparedas follows. 920 mg (2 mMol)7-butyryloxy-3-benzyloxycarbonylmethylamino-carbonyl-6-chlorocoumarinwere dissolved in 50 ml dioxane. 100 mg Palladium on carbon (10%) and100 μl acetic acid were added to the solution and the suspension stirredvigorously in a hydrogen atmosphere at ambient pressure. After theuptake of hydrogen seized the suspension was filtered. The productcontaining carbon was extracted five times with 25 ml boiling dioxane.The combined dioxane solutions were let to cool upon which the productprecipitated as a white powder. Reduction of the solvent to 20 mlprecipitates more product. The remaining dioxane solution is heated toboiling and heptane is added until the solution becomes cloudy. Theweights of the dried powders were 245 mg, 389 mg and 58 mg, totaling 692mg (1.88 mMol, 94%) of white product. ¹H NMR (dDMSO): δ 1.02 ppm (t, 3H,J 7.4 Hz, butyric methyl), δ 1.73 ppm (m, 2H, J₁ δ J₂=7.3 Hz, butyricmethylene), δ 2.70 ppm (t, 2H, J 0.67 ppm (s, 1H, coumarin), δ 8.35 ppm(s, 1H, coumarin), δ 8.90 ppm (s, 1H, coumarin), δ 9.00 ppm (t, 1H,J=5.6 Hz, amide).

Coupling of 7-Butyryloxy-3-carboxymethylaminocarbonyl-6-chlorocoumarinwith 7-amino-3′-chlorocephalosporanic acid benzhydryl ester was effectedas follows. 368 mg (1 mMol)7-Butyryloxy-3-carboxymethylaminocarbonyl-6-chlorocoumarin, 270 mghydroxybenztriazole hydrate and 415 mg (1 mMol) 7-amino-3′-chlorocephalosporanic acid benzhydryl ester were suspended in 40 ml dioxane-acetonitrile (1:1). 260 mg (1.25 mMol) dicyclohexylcarbodiimide in 5 mlacetonitrile were added and the suspension was stirred vigorously for 36hours. The precipitate was removed by filtration and the volume of thesolution reduced to 20 ml on the rotatory evaporator. 50 ml Toluene wasadded and the volume reduced to 30 ml. With stirring 50 ml heptane wasadded and the suspension chilled on ice. The precipitate was recoveredby filtration. It was redissolved in 10 ml chloroform and the remainingundissolved solids were filtered off. Addition of 2 volumes of heptaneprecipitated the title product which was collected and dried in vacuoand yielded 468 mg (0.64 mMol, 64%) off-white powder. ¹H NMR (CDCl₃): δ1.08 ppm (t, 3H, J 7.4 Hz, butyric methyl), δ 1.84 ppm (m, 2H, J₁ δ J₂7.4 Hz, butyric methylene), δ 2.66 ppm (t, 2H, J 7.4 Hz, butyricmethylene), δ 3.54 ppm (2d, 2H, J 18.3 Hz, cephalosporin C-2), δ 4.24ppm (2d, 2H, J 5.8 Hz, cephalosporin 3 methylene), δ 4.37 ppm (d, 2H, J3.8 Hz, glycine methylene), δ 5.02 ppm (d, 1H, J 4.9 Hz, cephalosporinC-6), δ 5.89 ppm (dd, 1H, J₁ 9.0 Hz, J₂ 5.0 Hz, cephalosporin C-7), δ6.96 ppm (s, 1H, benzhydryl), δ 7.30-7.45 ppm (m, 12H, phenyl, coumarin,amide), δ 7.79 ppm (s, 1H, coumarin), δ 8.84 ppm (s, 1H, coumarin), δ9.28 ppm (t, 1H, J 3.7 Hz, amide).

Coupling of the above product with 5-fluoresceinthiol was effected asfollows. 90 mg (0.2 mMol) 5-mercaptofluorescein diacetate disulfidedimer were dissolved in 10 ml chloroform and treated with 25 μl tributylphosphine and 25 μl water in an argon atmosphere. The solution was keptfor 2 hours at ambient temperature and was then added to a solution of20 mg sodium bicarbonate, 25 mg sodium iodide and 110 mg (0.15 mMol) ofthe above compound in 10 ml dimethylformamide. After 4 hours thesolvents were removed in vacuo and the residue triturated withdiethylether. The solid was dissolved in ethyl acetate-acetonitrile(1:1). After removal of the solvents the residue was triturated oncemore with diethylether yielding 157 mg (0.13 mMol, 88%) of acream-colored powder product.

A sample of the above compound was treated with a large access oftrifluoroacetic acid-anisole (1:1) at room temperature for 20 minutes.The reagents are removed in vacuo and the residue triturated with ether.High performance liquid chromatography of the solid in 45% aqueousacetonitrile containing 0.5% acetic acid gives a product in which thebutyrate and the diphenylmethyl esters have been cleaved. It waspurified by high performance liquid chromatography on a reverse phaseC₁₈-column using 45% aqueous acetonitrile containing 5% acetic acid asthe eluent.

Deprotection of the fluorescein acetates in compound 27 was accomplishedwith sodium bicarbonate in methanol (room temperature, 30 minutes) toprovide the fluorescent enzyme substrate CCF2. It was purified by highperformance liquid chromatography on a reverse phase C₁₈-column using35% aqueous acetonitrile containing 0.5% acetic acid as the eluent.

Stirring of compound 27 with excess acetoxymethyl bromide in drylutidine produced the menembrane permeable derivative of the substrate(CCF2/ac₂AM₂). It was purified by high performance liquid chromatographyon a reverse phase C₁₈ -column using 65% aqueous acetonitrile containing0.5% acetic acid as the eluent. CCF2/ac₂AM₂ is readily converted to CCF2in the cells' cytoplasm.

The donor and accepter dyes in substrate CCF2 do not stack. Thesubstrate is fully fluorescent in phosphate buffer and there is noformation of the “dark complex” (i.e., addition of methanol does notchange the fluorescence spectrum of CCF2, except for the effect ofdilution). This is due to the much smaller and more polar nature of the7-hydroxycoumarin compared to that of the xanthene dyes (eosin,rhodarnine, rhodol and resorufin).

The emission spectrum of compound CCF2 in 50 mmolar phosphate buffer pH7.0 before and after β-lactamase cleavage of the β-lactam ring. In theintact substrate, efficient energy transfer occurs from the7-hydroxycoumarin moiety to the fluorescein moiety. Excitation of thesubstrate at 405 nm results in fluorescence emission at 515 nm (green)from the acceptor dye fluorescein. The energy transfer is disrupted whenβ-lactamase cleaves the β-lactam ring, thereby severing the link betweenthe two dyes. Excitation of the products at 405 nm now results entirelyin donor fluorescence emission at 448 nm (blue). The fluorescenceemission from the donor moiety increases 25 fold upon β-lactam cleavage.The fluorescence at 515 nm is reduced by 3.5-fold, all of the remainingfluorescence originating from the 7-hydroxycoumarin as its emissionspectrum extends into the green. Twenty-five-fold quenching of the donorin the substrate is equivalent to an efficiency of fluorescence energytransfer of 96%. This large fluorescence change upon β-lactam cleavagecan readily be used to detect β-lactamase in the cytoplasm of livingmammalian cells.

The 7-hydroxycoumarin moiety in the cephalosporin was determined to havea fluorescence quantum efficiency in the absence of the acceptor of98-100%. This value was determined by comparing the integral of thecorrected fluorescence emission spectrum of the dye with that of asolution of 9-aminoacridine hydrochloride in water matched forabsorbance at the excitation wavelength. It follows that7-hydroxycoumarin is an ideal donor dye, as virtually every photonabsorbed by the dye undergoes fluorescence energy transfer to theacceptor.

Example 2 Use of a β Lactamase Substrate

Cells of the T-cell lymphoma line Jurkat were suspended in an isotonicsaline solution (Hank's balanced salt solution) containing approximately10¹² β-lactamase enzyme molecules per milliliter (approximately 1.7 nM;Penicillinase 205 TEM R⁺, from Sigma) and 1 mg/ml rhodamine conjugatedto dextran (40 kd) as a marker of loading. The suspension was passedthrough a syringe needle (30 gauge) four times. This causes transient,survivable disruptions of the cells' plasma membrane and allows entry oflabeled dextran and β-lactamase. Cells that had been successfullypermeabilized contained β-lactamase and were red fluorescent whenilluminated at the rhodamine excitation wavelength on a fluorescentmicroscope. The cells were incubated with 5 μM fluorogenic β-lactamasesubstrate, CCF2/ac₂AM₂, at room temperature for 30 minutes. Illuminationwith violet light (405 nm) revealed blue fluorescent and greenfluorescent cells. All cells that had taken up the markerrhodamine-dextran appeared fluorescent blue, while cells devoid theenzyme appeared fluorescent green.

Example 3 Use of a β Lactamase Substrate

Cells from cell lines of various mammalian origin were transientlytransfected with a plasmid containing the RTEM β-lactamase gene underthe control of a mammalian promoter. The gene encodes cytosolicβ-lactamase lacking any signal sequence and is listed as SEQ. D. 1.10 to48 hours after transfection cells were exposed to 5 micromolarCCF2/ac₂AM₂ for 1 to 6 hours. In all cases fluorescent blue cells weredetected on examination with a fluorescence microscope. Not a singleblue fluorescent cell was ever detected in non transfected controlcells. To quantitate the fluorescence measurements the cells were firstviewed through coumarin (450 DF 65) and then fluorescein (515 EFLP)emission filters and pictures were recorded with a charge couple devicecamera. The average pixel intensities of CCF2 loaded transfected cells(blue) and controls (green) at coumarin and fluorescein wavelength inCOS-7 (Table 2) and CHO (Table 3) cells are summarized; values for 4representative cells for each population are given. Thus, the substrateCCF2 revealed gene expression in single living mammalian cells.Substrate can be loaded using Pluronic formulations (see MolecularProbes Catalog) using polyethylene glycol. TABLE 1 COS-7 (origin: SV40transformed African green monkey kidney cells) coumarin emissionFluorescein emission Table of pixel intensities filter filter Blue cell#1 27 20 #2 34 23 #3 31 31 #4 22 33 Green cell #1 4 43 #2 4 42 #3 5 20#4 3 24

TABLE 2 CHO (origin: Chinese hamster ovary cells) coumarin emissionFluorescein emission Table of pixel intensities filter filter Blue cell#1 98 112 #2 70 113 #3 76 92 #4 56 67 Green cell #1 9 180 #2 9 102 #3 7101 #4 9 83

Example 4 Expression of Gα₁₅ and Gα₁₆ in Cells

This example illustrates that, although constitutive expression of Gα₁₅or Gα₁₆ at high levels is toxic to cells, expression of Gα₁₅ or Gα₁₆from a gene that is controlled by an inducible promoter, is tolerated bythe cells. For constitutive or inducible expression of Gα₁₅ or Gα₁₆, thegenes encoding each of these subunits were placed, separately, under thecontrol of a cytomegalovirus promoter in the plasmids pcDNA3Gα15 ,pcDNA3Gα16 (Vector pcDNA3 available from Invitrogen, Inc., Del Mar,Calif.), pdEF-BOSGα15 and pdE F-BOSGα16 (For pd BOSG, see Gossen andBujard, 1992, Proc. Natl. Acad. Sci. 89:5547-5551, also available fromClontech). To construct pdEF-BOSG 15 and pdEF-BOSG 16, sequencesencoding the G subunit were inserted into pdEFBOS at its EcoRI and NotIsites. The plasmid pdEFBOS was derived from pEFBOS by removing theHindIII fragment containing the SV40 Ori (see Mizushima and Nagata,1990, Nucl. Acids. Res. 18). Each of these plasmids was used totransfect COS-7 cells, according to conventional protocols, and eachplasmid carried a neo gene, which confers resistance to G418. As issummarized in Table 3, approximately 150 G418-resistant clones weregenerated, yet none of the clones was able to express a promiscuousG-protein. The ability of a cell to express a promiscuous G-protein wasdetermined by Western blot analysis using an antibody that binds apeptide having the amino acid sequence RPSVLARYLDEINLL (SEQ ID NO: 5)(Amatruda et al., 1991, Proc. Natl. Acad. Sci. 88:5587-5591). These datashow that constitutive expression of a promiscuous G-protein under thecontrol of a strong promoter is not tolerated by COS-7 cells.Constitutive expression of promiscuous G proteins at high levels maylead to constant accumulation of inositol phosphates or metabolites,which may be toxic to cells. TABLE 3 High-Level Constitutive Expressionof Promiscuous G-Proteins is not Tolerated G418-resistant Clonesexpressing Construct Selection clones picked Gα_(15/16) PcDNA3Gα15 G41853 0 PcDNA3Gα16 G418 48 0 pdEF-BOSGα15 G418 36 0 pdEF-BOSGα16 G418 19 0

The data summarized herein indicate that, although cells may nottolerate constitutive expression of promiscuous Gα proteins at highlevels, they can tolerate expression of promiscuous Gα proteins from aninducible promoter. In this case, the genes for Gα₁₅ and Gα₁₆ wereplaced under the control of a cytomegalovirus (CMV) promoter that wasoperably linked to a heptamerized tet operator (Gossen and Bujard, 1992,Proc. Natl. Acad. Sci. 89:5547-5551). The plasmid encoding Gα and; theplasmid encoding Gα₁₆ are identical, except sequences encoding Gα₁₆ inlieu of Gα₁₅. These plasmids were used to transfect COS-7 cells. Thesecells were co-transfected with a tetracyclin-dependent transactivator,rtTA, that is operably linked to a CMV promoter of a plasmid thatcarries a neomycin resistance gene (Gossen et al., 1995, Science268:1766-1769).

Expression of the Gα genes was induced by contacting the cells withdoxycyclin, a tetracyclin analog. In these experiments, the doxycyclinconcentration was 3 g/ml, although doxycyclin concentrations rangingfrom 0.01 to 10 μml can be used in order to regulate the level of geneexpression. Of 17 hygromycin-resistant clones that were analyzed, 2clones showed doxycyclin-dependent expression of Gα₁₅ or Gα₁₆ by Westernblot analysis as described above. FIG. 3 illustrates that, in thepresence of doxycyclin, expression of Gα₁₅ or Gα₁₆ is detectable as aband of approximately 43 kDa. This expression system provides low levelsof constitutive expression of Gα₁₅ or Gα₁₆ (e.g., less than 100 Gαproteins/cell), yet expression of the Gα protein is highly inducible. Upto 10,000 Gα proteins/cell are produced upon induction of geneexpression. As a control, COS-7 cells that lacked the Gα gene wereanalyzed, and Western blot analysis indicated that the control cells didnot express Gα₁₅ or Gα₁₆. In sum, these experiments demonstrate thatstable cells can be produced by employing an inducible promoter thatprovides (a) low levels of constitutive expression (i.e., producing lessthan approximately 100 Gα proteins/cell), and (b) high levels of inducedexpression (i.e., producing approximately 10,000 Gα proteins/cell).

Example 5 Detection of Gα Protein Activity by Detection of FluorescenceEmission

These examples demonstrate that activation of a Gα protein in a cell,and a change in expression of a reporter gene, can be detected by adetecting a change in fluorescence emission of a sample that includesthe cell. These examples employ Jurkat T lymphocytes that weretransfected with a genetic construct that expresses a reporter gene. Thegenetic construct includes a NFAT promoter, which is responsive toincreased calcium levels and protein kinase C activation that resultfrom activation of Gα protein. The NFAT promoter was operably linked toa β-lactamase reporter gene. To detect expression of the reporter gene,and thereby detect activation of Gα, the cells were contacted with theβ-lactamase substrate CCF2ac₂/AM₂ (described herein), and fluorescenceemission was detected according to previously described methods (Tsienet al., 1993, Trends in Cell Biology 3:242-245).

Two different compounds, ionomycin and phorbol myristate acetate (PMA),were used to optimize detection of expression of the reporter gene inthese examples. In the first example, the dose response to ionomycin wasmeasured. In this example, a set of samples of cells were contacted withPMA (at 3 nM) and the calcium ionophore ionomycin (at variousconcentrations, ranging from 0 to 3.0 μM). Ionomycin increases calciumlevels inside of the cells, and thereby increases the probability thatactivation of a G-protein, and a G-protein-mediated increase in calciumlevels, will activate expression of a reporter gene (e.g., a β-lactamasegene) that is operably linked to a calcium-responsive promoter (e.g., aNFAT promoter).

In practicing these methods, it is preferable to add the ionophore to alevel that is just below the threshold level for activation of thecalcium-responsive promoter (e.g., the NFAT promoter). Expression of thereporter gene then is activated by activation of the Gα protein, and thesubsequent rise in intracellular calcium levels. As is illustrated inFIG. 4A, fluorescence emission from a sample of the aforementioned cellscan be measured by FRET. In this example, fluorescence emission wasmeasured approximately 90 minutes after stimulation. Because thefluorogenic β-lactamase substrate undergoes a shift in fluorescenceemission, fluorescence emission is measured as an emission ratio(450/530) when exciting at 400 nm. This figure also illustrates that anionomycin concentration of approximately 0.3 μM is preferable forincreasing the intracellular calcium level to a level that is just belowthe threshold level for activation of the calcium-responsive promoter.

In a second example, the dose response of PMA required to stimulateNFAT-driven expression was measured. Although PMA does not, by itself,affect NFAT-regulated gene expression, it potentiates a cell's responseto an increase in calcium levels. In this example, a set of cells wastreated with ionomycin (at 1 μM ionomycin) and PMA (at variousconcentrations, ranging from 0 to 30 nM). As above, fluorescenceemission was measured 90 minutes after stimulation. As is illustrated inFIG. 4B, increasing concentrations of PMA increased fluorescenceemission from the cell sample. Thus, treating the cells with PMAenhances detection of expression of the reporter gene. This example alsoillustrates that a PMA concentration of approximately 3 nM is preferablefor enhancing detection of expression of a reporter gene.

Example 6 Monitoring Activation and Inhibition of GPCR Activity with anNFAT β-Lactamase Assay

This example demonstrates that activation of a GPCR (Gq receptorsubtype) can be detected with an NFAT β-lactamase assay, which is anexample of signal transduction detection system based on acalcium-responsive promoter transcription based assay. Stable cell line(production of described herein) containing Gq-type GPCR receptorexpresses β-lactamase in response to the addition of the agonist. TheGαq protein was endogenously expressed. This response is inhibited by anantagonist. Jurkat clones expressing NFAT-β1a were transfected withexpression vectors containing the Gq receptor and neomycin resistancegene (double transfection). The transfected population was neo-selectedand sorted by FACS for clones responding to the GPCR agonist. For theexperiments shown, cells were stimulated for three hours with theindicated ligands. Cells were then loaded with β-lactamase substrateCCF2/ac2AM for 1 hour, washed, dispensed into wells of a microtiterplate (100,000 cells/well) and the blue/green ratio was recorded by aplate reader.

FIG. 6 demonstrates a twenty-fold change in signal upon receptoractivation with an agonist (saturating dose 100 μM). A receptorantagonist (10 μM) completely inhibited the agonist activation of thereceptor.

Example 7 Monitoring Activation of GPCRs with a Calcium Dye (TransientlyTransfected Cells)

This example demonstrates that activation of GPCRs (Gs and Gi receptorsubtypes) can be detected with an intracellular calcium indicatortransiently transfected cells, which is an example of a signaltransduction detection system based on changes in intracellular ions.The cells used were transiently transfected with two constructs. 4×10⁵CHO-K1 cells were seeded on 35 mm petri dishes one day beforetransfection. 5 μg plasmid DNA and 12 μl lipofectamine were added foreach dish using the stand method. In some cases, pBluescripts (−) or KSplasmid was used to keep the amount of DNA consistent between eachtransfection. 20 h later, the cells were stained with 10 mM Fura-PE3(Molecular Probe) for 3 h. Imaging analysis of calcium was performed tomeasure the [Ca²⁺] signal mediated by the agonists addition.

Following imaging data show that the promiscuous Gα 16 couples aGs-receptor and a Gi-receptor in CHO-K1 cells following transienttransfections. The data also show that promiscuous Gα protein can changethe effector downstream of the GPCR. Panel A: 60 seconds after startingof the experiment, 10 μM agonist solution was added to the cellstransfected by pCIS/Gα 16 (CMV promoter) and Gs-receptor (CMV promoter)expression plasrnids. Panel B: 60 seconds after starting of theexperiment, 10 μM agonist solution was added to the cells transfected bypCIS/Gα 16 alone. Panel C: 60 seconds after starting of the experiment,10 μM agonist solution was added to the cells transfected by Gs receptorexpression plasmid alone. Panel D: 60 seconds after starting of theexperiment, 10 μM agonist solution was added to the cells transfected bypCIS/Gα16 and Gi-receptor expression plasnids. Panel E: 60 seconds afterstarting of the experiment, 10 μM agonist solution was added to thecells transfected by pCIS/Gα 16 alone. Panel F: 60 seconds afterstarting of the experiment, 10 μM agonist solution was added to thecells transfected by Gi-receptor expression plasmid alone.

Example 8 Monitoring Activation of a GPCR with a Calcium Dye (StablyTransfected Cells)

This example demonstrates that activation of a GPCR (Gs receptorsubtype) can be detected with an intracellular calcium indicator instably transfected cells, which is an example of a signal transductiondetection system based on changes in intracellular ions. The cells usedwere transiently stably with two constructs. Although many cells do nottolerate stable expression of promiscuous Gα protein, such as describedherein, surprisingly even cells thought not to tolerate stableexpression of promiscuous Gα protein can be sorted using a signaltransduction detection system. Such sorting can be performed with a highthroughput sorting system, such as a FACS or 96 well imaging system.Typically, the frequency of usable stable cells is about 1 to 2 percentof those cells screened. Functional assay selection of promiscuous Gαprotein/GPCR double transfected cells is a preferred method ofidentifying cells that either tolerate, or express the proper amounts,of promiscuous Gα protein and a GPCR.

Stable CHO-K1 cell lines expressing Gα 15-Hyg alone, the Gs-receptor-Neoalone and both the Gα15 (CMV promoter) and the Gs receptor (CMVpromoter) (double transfection), were generated. 48 h after transfection(described herein for the method of lipofectamine-mediatedtransfection), media containing Hygromycin (0.5 mg/ml), Neomycin (1mg/ml) or both were added on to the cells to select the stabletransformants. 12-15 days after selection, the stable clones wereexamined using the calcium imaging assays.

The following imaging data show that the promiscuous Gα15 couples aGs-receptor in CHO-K1 cells following stable cell line generation. Thedata also show that promiscuous Gα protein can change the effectordownstream of the GPCR Panel A: Calcium imaging of the Gα15/Gs-receptordual stable clone-2. 10 μM agonist was added 40 seconds after thestarting of the experiment. Panel B: Calcium imaging of the Gs-receptorstable clone-2. 10 μM agonist was added 40 seconds after the starting ofthe experiment. Panel C: Calcium imaging of the Gα5 stable clone-H. 10μM agonist was added 40 seconds after the starting of the experiment.

Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

REFERENCES

-   Bram, R. J. and G. R. Crabtree. (1994) Nature 371:355-358.-   Offermanns, S. and M. I. Simon. (1995) J. Biol. Chem. 270(25):    15175-15180.-   Fiering, S., Northrop, J. P., Nolan, G. P., Mattila, P. S.,    Crabtree, G. R., and Herzenberg, L. A. (1990) Genes Dev. 4,    1823-1834.-   Flanagan, W. F., Corthesy, B., Bram, R. J. and    Crabtree, G. R. (1991) Nature 352:803-807.-   Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W.,    and H. Bujard (1995) Science 268: 1766-1769.-   Grinkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem.    260:3440-3450.-   Neer, E. J. (1995). Cell, 80:249-257.-   Negulescu, P. A., Shastri, N., Cahalan, Michael D. (1994). Proc.    Nat. Acad Sci. 91:2873-2877.-   Sternweis, P. C. and A. V. Smrcka (1992) Trends Biochem. Sci.    17:502-506.-   Thastrup, O., Cullen, P. J., Drobak, B. K., Hanley, M. R., and    Dawson, A. P. (1990) Proc. Natl. Acad. Sci. 87:2466-2470.-   Tsien, R. Y., Backsai, B. J., and Adams, S. R. (1993) Trends Cell    Biol. 3:242-245.

All publications, including patent documents and scientific articles,referred to in this application are incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication were individually incorporated by reference.

Sequence ID Listing

Nucleotide and Amino Acid Sequences of Gα15

(SEQ ID NO: 2 and SEQ ID NO: 4, respectively)          9          18          27          36          45          54ATG GCC CGG TCC CTG ACT TGG GGC TGC TGT CCC TGG TGC CTG ACA GAG GAG GAG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Met Ala Arg Ser Leu Thr Trp Gly Cys Cys Pro Trp Cys Leu Thr Glu Glu Glu         63          72          81          90          99         108AAG ACT GCC GCC AGA ATC GAC CAG GAG ATC AAC AGG ATT TTG TTG GAA CAG AAA--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Lys Thr Ala Ala Arg Ile Asp Gln Glu Ile Asn Arg Ile Leu Leu Glu Gln Lys        117         126         135         144         153         162AAA CAA GAG CGC GAG GAA TTG AAA CTC CTG CTG TTG GGG CCT GGT GAG AGC GGG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Lys Gln Glu Arg Glu Glu Leu Lys Leu Leu Leu Leu Gly Pro Gly Glu Ser Gly        171         180         189         198         207         216AAG AGT ACG TTC ATC AAG CAG ATG CGC ATC ATT CAC GGT GTG GGC TAC TCG GAG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Lys Ser Thr Phe Ile Lys Gln Met Arg Ile Ile His Gly Val Gly Tyr Ser Glu        225         234         243         252         261         270GAG GAC CGC AGA GCC TTC CGG CTG CTC ATC TAC CAG AAC ATC TTC GTC TCC ATG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Glu Asp Arg Arg Ala Phe Arg Leu Leu Ile Tyr Gln Asn Ile Phe Val Ser Met        279         288         297         306         315         324CAG GCC ATG ATA GAT GCG ATG GAC CGG CTG CAG ATC CCC TTC AGC AGG CCT GAC--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Gln Ala Met Ile Asp Ala Met Asp Arg Leu Gln Ile Pro Phe Ser Arg Pro Asp        333         342         351         360         369         378AGC AAG CAG CAC GCC AGC CTA GTG ATG ACC CAG GAC CCC TAT AAA GTG AGC ACA--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Ser Lys Gln His Ala Ser Leu Val Met Thr Gln Asp Pro Tyr Lys Val Ser Thr        387         396         405         414         423         432TTC GAG AAG CCA TAT GCA GTG GCC ATG CAG TAC CTG TGG CGG GAC GCG GGC ATC--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Phe Glu Lys Pro Tyr Ala Val Ala Met Gln Tyr Leu Trp Arg Asp Ala Gly Ile        441         450         459         468         477         486CGT GCA TGC TAC GAG CGA AGG CGT GAA TTC CAC CTT CTG GAC TCC GCG GTG TAT--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Arg Ala Cys Tyr Glu Arg Arg Arg Glu Phe His Leu Leu Asp Ser Ala Val Tyr        495         504         513         522         531         540TAC CTG TCA CAC CTG GAG CGC ATA TCA GAG GAC AGC TAC ATC CCC ACT GCG CAA--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Tyr Leu Ser His Leu Glu Arg Ile Ser Glu Asp Ser Tyr Ile Pro Thr Ala Gln        549         558         567         576         585         594GAC GTG CTG CGC AGT CGC ATG CCC ACC ACA GGC ATC AAT GAG TAC TGC TTC TCC--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Asp Val Leu Arg Ser Arg Met Pro Thr Thr Gly Ile Asn Glu Tyr Cys Phe Ser        603         612         621         630         639         648GTG AAG AAA ACC AAA CTG CGC ATC GTG GAT GTT GGT GGC CAG AGG TCA GAG CGT--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Val Lys Lys Thr Lys Leu Arg Ile Val Asp Val Gly Gly Gln Arg Ser Glu Arg        657         666         675         684         693         702AGG AAA TGG ATT CAC TGT TTC GAG AAC GTG ATT GCC CTC ATC TAC CTG GCC TCC--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Arg Lys Trp Ile His Cys Phe Glu Asn Val Ile Ala Leu Ile Tyr Leu Ala Ser        711         720         729         738         747         756CTG AGC GAG TAT GAC CAG TGC CTA GAG GAG AAC GAT CAG GAG AAC CGC ATG GAG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Leu Ser Glu Tyr Asp Gln Cys Leu Glu Glu Asn Asp Gln Glu Asn Arg Met Glu        765         774         783         792         801         810GAG AGT CTC GCT CTG TTC AGC ACG ATC CTA GAG CTG CCC TGG rrC AAG AGC ACC--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Glu Ser Leu Ala Leu Phe Ser Thr Ile Leu Glu Leu Pro Trp Phe Lys Ser Thr        819         828         837         846         855         864TCG GTC ATC CTC TTC CTC AAC AAG ACG GAC ATC CTG GAA GAT AAG ATT CAC ACC--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Ser Val Ile Leu Phe Leu Asn Lys Thr Asp Ile Leu Glu Asp Lys Ile His Thr        873         882         891         900         909         918TCC CAC CTG GCC ACA TAC TTC CCC AGC TTC CAG GGA CCC CGG CGA GAC GCA GAG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Ser His Leu Ala Thr Tyr Phe Pro Ser Phe Gln Gly Pro Arg Arg Asp Ala Glu        927         936         945         954         963         972GCC GCC AAG AGC TTC ATC TTG GAC ATG TAT GCG CGC GTG TAC GCG AGC TGC GCA--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Ala Ala Lys Ser Phe Ile Leu Asp Met Tyr Ala Arg Val Tyr Ala Ser Cys Ala        981         990         999        1008        1017        1026GAG CCC CAG GAC GGT GGC AGG AAA GGC TCC CGC GCG CGC CGC TTC TTC GCA CAC--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Glu Pro Gln Asp Gly Gly Arg Lys Gly Ser Arg Ala Arg Arg Phe Phe Ala His       1035        1044        1053        1062        1071        1080TTC ACC TGT GCC ACG GAC ACG CAA AGC GTC CGC AGC GTG TTC AAG GAC GTG CGG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Phe Thr Cys Ala Thr Asp Thr Gln Ser Val Arg Ser Val Phe Lys Asp Val Arg       1089        1098        1107        1116        1125 GAC TCG GTGCTG GCC CGG TAC CTG GAC GAG ATC AAC CTG CTG TGA --- --- --- --- --- ------ --- --- --- --- --- --- --- --- Asp Ser Val Leu Ala Arg Tyr Leu AspGlu Ile Asn Leu Leu ***

Nucleotide and Amino Acid Sequences of G 16

(SEQ ID NO: 1 and SEQ ID NO: 3, respectively)          9          18          27          36          45          54ATG GCC CGC TCG CTG ACC TGG CGC TGC TGC CCC TGG TGC CTG ACG GAG GAT GAG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Met Ala Arg Ser Leu Thr Trp Arg Cys Cys Pro Trp Cys Leu Thr Glu Asp Glu         63          72          81          90          99         108AAG GCC GCC GCC CGG GTG GAC CAG GAG ATC AAC AGG ATC CTC TTG GAG CAG AAG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Lys Ala Ala Ala Arg Val Asp Gln Glu Ile Asn Arg Ile Leu Leu Glu Gln Lys        117         126         135         144         153         162AAG CAG GAC CGC GGG GAG CTG AAG CTG CTG CTT TTG GGC CCA GGC GAG AGC GGG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Lys Gln Asp Arg Gly Glu Leu Lys Leu Leu Leu Leu Gly Pro Gly Glu Ser Gly        171         180         189         198         207         216AAG AGC ACC TTC ATC AAG CAG ATG CGG ATC ATC CAC GGC GCC GGC TAC TCG GAG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Lys Ser Thr Phe Ile Lys Gln Met Arg Ile Ile His Gly Ala Gly Tyr Ser Glu        225         234         243         252         261         270GAG GAG CGC AAG GGC TTC CGG CCC CTG GTC TAC CAG AAC ATC TTC GTG TCC ATG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Glu Glu Arg Lys Gly Phe Arg Pro Leu Val Tyr Gln Asn Ile Phe Val Ser Met        279         288         297         306         315         324CGG GCC ATG ATC GAG GCC ATG GAG CGG CTG CAG ATT CCA TTC AGC AGG CCC GAG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Arg Ala Met Ile Glu Ala Met Glu Arg Leu Gln Ile Pro Phe Ser Arg Pro Glu        333         342         351         360         369         378AGC AAG CAC CAC GCT AGC CTG GTC ATG AGC CAG GAC CCC TAT AAA GTG ACC ACG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Ser Lys His His Ala Ser Leu Val Met Ser Gln Asp Pro Tyr Lys Val Thr Thr        387         396         405         414         423         432TTT GAG AAG CGC TAC GCT GCG 0CC ATG CAG TGG CTG TGG AGG GAT GCC GGC ATC--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Phe Glu Lys Arg Tyr Ala Ala Ala Met Gln Trp Leu Trp Arg Asp Ala Gly Ile        441         450         459         468         477         486CGG GCC TGC TAT GAG CGT CGG CGG GAA TTC CAC CTG CTC GAT TCA GCC GTG TAC--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Arg Ala Cys Tyr Glu Arg Arg Arg Glu Phe His Leu Leu Asp Ser Ala Val Tyr        495         504         513         522         531         540TAC CTG TCC CAC CTG GAG CGC ATC ACC GAG GAG GGC TAC GTC CCC ACA GCT CAG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Tyr Leu Ser His Leu Glu Arg Ile Thr Glu Glu Gly Tyr Val Pro Thr Ala Gln        549         558         567         576         585         594GAC GTG CTC CGC AGC CGC ATG CCC ACC ACT GGC ATC AAC GAG TAC TGC TTC TCC--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Asp Val Leu Arg Ser Arg Met Pro Thr Thr Gly Ile Asn Glu Tyr Cys Phe Ser        603         612         621         630         639         648GTG CAG AAA ACC AAC CTG CGG ATC GTG GAC GTC 000 GGC CAG AAG TCA GAG CGT--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Val Gln Lys Thr Asn Leu Arg Ile Val Asp Val Gly Gly Gln Lys Ser Glu Arg        657         666         675         684         693         702AAG AAA TGG ATC CAT TGT TTC GAG AAC GTG ATC GCC CTC ATC TAC CTG GCC TCA--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Lys Lys Trp Ile His Cys Phe Glu Asn Val Ile Ala Leu Ile Tyr Leu Ala Ser        711         720         729         738         747         756CTG AGT GAA TAC GAC CAG TGC CTG GAG GAG AAC AAC CAG GAG AAC CGC ATG AAG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Leu Ser Glu Tyr Asp Gln Cys Leu Glu Glu Asn Asn Gln Glu Asn Arg Met Lys        765         774         783         792         801         810GAG AGC CTC GCA TTG TTT GGG ACT ATG CTG GAA CTA CCC TGG TTC AAA AGC ACA--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Glu Ser Leu Ala Leu Phe Gly Thr Ile Leu Glu Leu Pro Trp Phe Lys Ser Thr        819         828         837         846         855         864TGC GTC ATC CTC TTT CTC AAC AAA ACC GAC ATC CTG GAG GAG AAA ATC CCC ACC--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Ser Val Ile Leu Phe Leu Asn Lys Thr Asp Ile Leu Glu Glu Lys Ile Pro Thr        873         882         891         900         909         918TCC CAC CTG GCT ACC TAT TTC CCC AGT TTC CAG GGC CCT AAG CAG GAT GCT GAG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Ser His Leu Ala Thr Tyr Phe Pro Ser Phe Gln Gly Pro Lys Gln Asp Ala Glu        927         936         945         954         963         972GCA GCC AAG AGG TTC ATC CTG GAC ATG TAC ACG AGG ATG TAC ACC GGG TGC GTG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Ala Ala Lys Arg Phe Ile Leu Asp Met Tyr Thr Arg Met Tyr Thr Gly Cys Val        981         990         999        1008        1017        1026GAC GGC CCC GAG GGC AGC AAG AAG GGC GCA CGA TCC CGA CGC CTT TTC AGC CAC--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Asp Gly Pro Glu Gly Ser Lys Lys Gly Ala Arg Ser Arg Arg Leu Phe Ser His       1035        1044        1053        1062        1071        1080TAC ACA TGT GCC ACA GAC ACA CAG AAC ATC CGC AAG GTC TTC AAG GAC GTG CGG--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---Tyr Thr Cys Ala Thr Asp Thr Gln Asn Ile Arg Lys Val Phe Lys Asp Val Arg       1089        1098        1107        1116        1125 GAC TCG GTGCTC GCC CGC TAC CTG GAC GAG ATC AAC CTG CTG TGA --- --- --- --- --- ------ --- --- --- --- --- --- --- --- Asp Ser Val Leu Ala Arg Tyr Leu AspGlu Ile Asn Leu Leu ***

1. A stable, isolated cell comprising a construct comprising promoteroperably linked to a polynucleotide encoding a polypeptide having abiological activity of a promiscuous Gα protein.
 2. The cell of claim 1,wherein said promoter is an inducible promoter.
 3. The cell of claim 2,wherein said construct permits expression in a mammalian cell.
 4. Thecell of claim 1, wherein said polynucleotide has a nucleotide sequencewith at least 70% sequence identity to a nucleotide sequence selectedfrom the group consisting of the nucleotide sequence of Gα₁₆ (SEQ ID NO:1), and the nucleotide sequence of Gα₁₅ (SEQ ID NO: 2). 5.-7. (canceled)8. The cell of claim 1, further comprising a second construct comprisinga reporter gene operably linked to a second promoter, wherein saidsecond promoter is modulated by a promiscuous Gα protein.
 9. The cell ofclaim 8, wherein the reporter gene encodes a reporter selected from thegroup consisting of luciferase, green fluorescent protein,chloramphenicol acetyl transferase, β-galactosidase, alkalinephosphatase, β-lactamase, and human growth hormone. 10.-11. (canceled)12. The cell of claim 8, wherein the reporter gene encodes β-lactamase.13. The cell of claim 8, wherein said second promoter comprises aprotein kinase C-responsive promoter. 14.-26. (canceled)
 27. A method ofidentifying a GPCR for a given ligand, the method comprising: (i)expressing a putative GPCR in a cell of claim 1; (ii) contacting saidcell with a ligand; and (iii) detecting reporter gene expression. 28.The method of claim 27, wherein said detecting comprises fluorescencedetection.
 29. The method of claim 28, further comprising contactingsaid cell with a reporter gene substrate.
 30. The method of claim 27,further comprising contacting said cell with a compound that increasescalcium levels inside said cell.
 31. The method of claim 30, whereinsaid compound is selected from the group consisting of ionomycin andthapsigargin.
 32. The method of claim 30, further comprising contactingsaid cell with phorbol myristate acetate or an analog thereof.
 33. Amethod of a identifying of a ligand for a given GPCR, the methodcomprising: a) expressing a GPCR in a cell of claim 1; b) contactingsaid cell with a test chemical; and c) detecting a signal with a signaltransduction detection system. 34.-36. (canceled)
 37. The method ofclaim 33, wherein said detecting comprises reporter gene detection. 38.A method for identifying a modulator of signal transduction in a cell,the method comprising: a) contacting a cell of claim 1 with a testchemical; b) contacting said cell with a ligand that, in the absence ofthe test chemical, activates signal transduction in said cell, and c)detecting a signal with a signal transduction detection system. 39.-42.(canceled)
 43. A kit comprising assay reagents and a containercontaining a cell of claim
 1. 44.-62. (canceled)